1. Field of the Invention
The present invention relates to novel phosphoric acid derivatives and a method for determination of acid phosphatase activity using the same. According to the present invention, acid phosphatase activity can be accurately determined in a simple way, and the present invention is extremely useful as an assay for determining acid phosphatase in the fields of medical treatment and laboratory procedures.
2. Related Art Statement
Acid phosphatase (hereafter referred to as Acp) is an enzyme that hydrolyzes a phosphoric acid monoester under acidic conditions (pH of 4 to 6). In patients with prostatic cancer, breast cancer accompanied by bone metastasis or bone diseases, and liver and renal diseases, an increase of Acp in serum or urine is noted; and, attention has been brought to determination of Acp as a tumor marker.
So far, methods using various synthetic substrates as shown below have been reported for assaying acid phosphatase activity. Some of them have been practically applied to ordinary laboratory procedures.
(a) Method using .beta.-glycerophosphoric acid as substrate [Bodansky, A.: J. Biol. Chem., 101, 93 (1933)]:
Hydrolysis of .beta.-glycerophosphoric acid by Acp gives glycerine and inorganic phosphorus. The inorganic phosphorus is allowed to produce a color and the color is measured.
(b) Method using p-nitrophenylphosphoric acid as substrate [Hudson, P.B.: J. Urol., 58, 89 (1947)]:
Hydrolysis of p-nitrophenylphosphoric acid by Acp gives p-nitrophenol, which is reacted with an alkali to form a color and the color is measured.
(c) Method using phenylphosphoric acid as substrate:
There are a method which comprising hydrolyzing phenylphosphoric acid with Acp and reacting the resulting phenol with Folin-Ciocalten reagent [King, E.J., Armstrong, A.R.: Canad. Med. Assoc. J., 31, 376 (1934)] and a method which comprises hydrolyzing phenylphosphoric acid with Acp, oxidatively condensing the resulting phenol with 4- aminoantipyrine and measuring the formed quinone of a red color [Kind, P.R.N., King, E.J.: J. Clin. Path., 7, 322 (1954)].
(d) Method using naphthylphosphoric acid as substrate [Hillman, G.: Z. Klin. Chem., Klin. U. Biochem., 9, 237 (1971)]:
Naphthol formed by hydrolysis with Acp is reacted with Fast Red TR to form an azo dye and the azo dye is colorimetrically determined.
(e) Method using 2,6-dichloro-4-nitrophenylphosphoric acid as substrate [Teshima, S., Hayashi, Y., Ando, M.: Clin. Chim. Acta., 168, 231 (1987)]:
A yellow hue of 2,6-dichloro-4-nitrophenol formed by hydrolysis with Acp is colorimetrically determined at 400nm.
These assay methods involve various problems and the problems are causes for inaccuracy of measurement data. For example, in method (a), inorganic phosphorus is contained in normal serum and hence, the inorganic phosphorus in serum to be tested must be previously assayed. Furthermore, complicated operations are required and such are problems in practical application. In method (b), after the enzymatic reaction in an acidic region which is the optimum pH of Acp, the system should be rendered alkaline with sodium hydroxide aqueous solution, etc., otherwise p-nitrophenol as a chromophore does not form a color and it is thus impossible to perform rate assay. Furthermore, the wavelength at 405 nm to be measured corresponds to a slope of U.V. spectrum of the chromophore and is seriously affected by bilirubin in serum, resulting in a cause for erroneous measurement data. Method (c) requires a color-forming reaction of phenol formed by hydrolysis with Acp as in method (b). It is thus impossible to conduct rate assay. In addition, the color formation between phenol and 4-aminoantipyrine is also unstable and this is another cause for error. In method (d), Fast Red TR which is reacted with free naphthol is unstable. Furthermore, the method involves a large lag time in the reaction which causes an error in measurement data, although the method may also be applicable to rate assay and to an automated analysis device.
Method (e) enables one to perform rate assay without requiring any color-forming reaction and is also applicable to automated analysis instruments. However, the method is liable to be affected by bilirubin or hemoglobin in serum since the wavelength for the measurement is close to 400 nm. In addition, the substrate itself is unstable in an aqueous solution and causes spontaneous hydrolysis.
As described above, the conventional methods for determining Acp activity involves various defects and the defects cause incorrect measurement data. Therefore, these methods are practically disadvantageous.